The present invention relates to a process of recovering intracellular proteins and other molecules from a cell.
It is desirable to lyse cells grown as production hosts containing a protein or other molecule of interest to recover any desired intracellularly produced product. Conventional ways to kill and lyse such cells include the use of heat (U.S. Pat. No. 4,601,986 to Wegner, et al.), osmotic pressure (U.S. Pat. No. 4,299,858 to Aubert, et al), enzymes which break down the cell walls or membranes (U.S. Pat. No. 3,816,260 to Sugiyama, U.S. Pat. No. 3,890,198 to Kobayashi, et al. and U.S. Pat. No. 3,917,510 to Kitamura, et al.) and mechanical disruption of the cell wall by, for example, high pressure homogenization. The disclosures of the above patents are incorporated herein by reference.
Also, detergents have been utilized to lyse the cell wall. For example, yeast protein extraction reagent (Y-PER(copyright)), sold by Pierce Chemical Company, contains a detergent to provide a gentle means of cell lysis that is not detrimental to the protein of interest. However, Y-PER(copyright) is intended to be used as a laboratory bench reagent, not as a reagent useful for the large scale production of proteins, and is costly. For these reasons, Y-PER(copyright) has not gained acceptance as a useful reagent for the large scale production of recombinant protein from host cells.
There is a need in the art for a process that can be used to easily cause the release of proteins from host cells without harming the desired protein and with a minimum of process steps. The method of cell lysis should not directly or indirectly lead to denaturation of the desired product and the method should be consistent with subsequent processing requirements and with large scale production.
The present invention relates to a process of releasing a protein, recombinant or otherwise, from a cell. The process of the present invention involves contacting a host cell containing a protein of interest with a solution comprising one or more detergents and one or more reducing agents. The addition of one or more reducing agents facilitates the recovery of proteins in their native conformations. The methods of the invention are particularly suitable to large scale production of recombinant products. The methods of the invention comprise four basic steps: adjustment of bulk solution conditions to achieve a permissive environment, contact of cells with a reducing agent either before, during or after contact of the cells with certain charge-modified hydrocarbons, and finally clarification of the extract to produce a fraction suitable for formulation or further processing. Typically, the reducing agent and the detergent are added sequentially in any order, resulting in the concurrent exposure of the cells to the reducing agent and the detergent.
In a particular embodiment, the one or more detergents are amphipathic, charged amines or amine oxides coupled to hydrocarbon chains of varying lengths. In a preferred embodiment, the one or more detergents used are selected from the group consisting of, tributylphosphate, dimethyldecylamine, dimethyltridecylamine, dimethylundecylamine, dimethyldidecylamine, dimethytetradecylamine, dimethylhexadecylamine, dimethyldecylamineoxide, dimethylundecylamineoxide, dimethyldidecylamineoxide, dimethytetradecylamineoxide and dimethyltridecylamineoxide. Preferably, the detergent is not dimethyltridecylamine.
Detergents may be used at concentrations ranging from 0.01% up to their solubility limit. Preferably, the concentration of the detergents ranges from 0.05% to 5%, 0.1% to 2%, or is approximately 0.5% of the total solution. When added to cells suspended in buffer, the detergent is preferably at a higher concentration than the final concentration at which the cells are lysed. Preferably, the detergent is at least at a 2 fold, 5 fold, 10 fold or 100 fold higher concentration.
In a particular embodiment, the one or more reducing agents are agents are those reducing agents that reduce disulfide bonds and/or maintain sulfhydryl residues in the reduced form. Any such reducing agent or agents may be used. In a preferred embodiment, the one or more reducing agents used are selected from the group consisting of, Dithiothreitol (DTT); Dithioerythiritol (DTE); Cysteine (Cys) and Tris 2-carboxyethyphosphine (TCEP).
Reducing agents may be used at concentrations ranging from 0.1 mM to 100 mM, 1 mM to 25 mM, 2 mM to 10 mM, or about 5 mM. When added to cells suspended in buffer, the reducing agent is preferably at a higher concentration than the final concentration at which the cells are lysed. Preferably, the detergent is at least at a 2 fold, 5 fold, 10 fold or 100 fold higher concentration.
In addition to the one or more detergents and reducing agents, in a preferred embodiment, the cells are also contacted with glycerol. Preferably, the glycerol concentration is at least 0.6%, or ranges from 0.6% to 20%, 0.6% to 15%, 0.6% to 12%, 0.6% to 6%, 0.6% to 3%, or 0.6% to 1%.
The pH of the solution can range from pH 2 to pH 12. Preferably, the solution is at a pH ranging from pH 5.0 up to pH 8.0. More preferably, the pH ranges from pH 5.5 to 7.4, from pH 6 to 7.4, from pH 7.0 to 7.4, or is approximately pH 7.3.
The recovery of protein from the cells with the solution of the invention can be carried out at a temperature of from about 2xc2x0 C. to about 50xc2x0 C. Preferably, the temperature is from about 2xc2x0 C. to about 30xc2x0 C., about 2xc2x0 C. to about 20xc2x0 C., about 2xc2x0 C. to about 10xc2x0 C., about 3xc2x0 C. to about 10xc2x0 C., about 4xc2x0 C., about 25xc2x0 C., or at room temperature.
The xe2x80x9chost cellsxe2x80x9d are cells containing a protein of interest. A xe2x80x9cprotein of interestxe2x80x9d is any protein present in a host cell that one desires to release from the host cell and, optionally, subsequently isolate or purify. Preferably, the protein of interest is a recombinant protein. In a preferred embodiment, the protein of interest has a molecular weight of less than 100 kD. In a further preferred embodiment, the protein of interest has a molecular weight of between 5 and 75 kD, preferably about 50 kD. The host cells may be of any type, preferably mammalian, bacterial, yeast, fungal, plant, avian, or reptilian. Most preferably, the host cells are yeast cells. In a particular embodiment, the yeast cells are of the species Pichia pastoris. 
In addition to releasing proteins from host cells, the composition of the invention may be used to release other molecules from host cells including nucleic acids, lipids, vitamins, small molecules and other cell, cytosolic, or organelle derived molecules or molecular complexes.